Proteomic analysis of antibody response in a case of laboratory-acquired infection with Francisella tularensis subsp. tularensis.

Immunoproteomic analysis was applied to study the immunoreactivity of serum samples collected at different time points from a laboratory assistant accidentally infected with highly virulent strain of Francisella tularensis subsp. tularensis. Immunoblotting showed that the spectrum of F. tularensis antigens recognized specifically by immune sera remained with the exception for 1 antigen stable for up to 16 years after infection. Using immunoproteomics approach 10 immunoreactive antigens were successfully identified. Several new immunogenic F. tularensis proteins were described for the first time.

Francisella tularensis strain LVS resides in MHC II-positive autophagic vacuoles in macrophages.

The Francisella tularensis strain LVS phagosome disintegrates during the first few hours after bacterial entry and microbes are released to the cytosol. Within 12 h both rapid multiplication of microbes and a steep increase of apoptosis of infected macrophages occur. We searched for signals involved in the death of macrophages and detected molecules associated with the autophagy machinery cathepsin D, PTEN, p53 and LC3, whose levels or modification were influenced by ongoing in vitro tularemic infection. The sequestration of cytoplasmic F. tularensis LVS into autophagosomes was confirmed by co-localization of the LVS strain containing vacuoles with LC3 (an autophagosomal marker). We also demonstrated the presence of MHC II antigens in these autophagosomes, indicating that they might act as a source of endogenous tularemic antigens for presentation to CD4+ T lymphocytes.

Initial peptide mass fingerprinting analysis of proteins obtained by lysis of Coxiella burnetii cells.

Whereas the complete genome of Coxiella burnetii (C.b.), the etiological agent of Q-fever, has recently been published (Seshadri et al., Proc. Natl. Acad. Sci. USA. 100, 5455-5460, 2003), the C.b. proteome is still under study. Using the bioinformatic approach, we found in total 309 proteins on two dimensional electroctrophoretic images of C.b. whole cell lysates. Eighteen major protein species were subjected to peptide mass fingerprinting and identified as the products of 6 known open reading frames (ORFs): the chaperone DnaK (heat shock 70 K protein), chaperonin 60 K (GroEL protein, heat shock protein B), DnaJ-like protein djlA (mucoidy activation protein mucZ), elongation factor Ts (EF-Ts), ribosomal protein L7/L12, and chaperonin 10 K (GroES protein, heat shock protein A).

Tick salivary gland extract accelerates proliferation of Francisella tularensis in the host.

Accelerated proliferation of the tick-borne bacterial pathogen Francisella tularensis was demonstrated in mice when the bacterium was injected together with salivary gland extract from Ixodes ricinus ticks. A significant increase in the numbers of bacteria was recorded in the dermal site of infection,the draining lymph nodes, and the spleen. Analysis of the expression of cytokine messenger ribonucleic acids showed polarization toward a Th2 profile. Salivary gland extract-mediated suppression of interleukin-12 and interferon-gamma, the cytokines required for the expression of the protective immunity against tularemic infection, apparently contributed to the decreased resistance against this tick-transmitted pathogen.

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Natural resistance to intracellular pathogens: modulation of macrophage signal transduction related to the expression of the Bcg locus.

In mice, the Bcg/Nramp1 gene of the chromosome 1 has been implicated in natural resistance or susceptibility to infection with several intramacrophage microorganisms. Functional studies of Bcg/Nramp1 congenic macrophages have shown that this gene has many pleiotropic effects on macrophage activation and function. Although a specific role of Bcg/Nramp1 in the control of pleiotropic effects has not been defined yet, several observations propose unifying hypothesis for its complex role: metal ion transport is the primary function of the Bcg/Nramp1 gene, the availability of metal ions as cofactors for many proteins results from this primary function and, in turn, the effect on signal transduction results from ion-regulated expression of cellular proteins and their functions. In the present study, we examined the possible alterations in signal transduction pathways related to different allelic expression of the Bcg locus in B10R (Bcgr/Nramp1s) and B10S (Bcgs/Nramp1r) macrophages. We have utilized 1-DE and 2-DE immunoblot analyses and investigated phosphorylation of proteins using either anti-phosphotyrosine antibody or antibodies recognizing specific phospho-forms of signaling proteins. In the basal state, B10R macrophages had a superior ability to phosphorylate p38 mitogen-activated protein kinase (MAPK) and manganese superoxide dismutase. B10S counterparts were characterized by increased phosphorylation of Erk1/Erk2 MAPKs. The activation of macrophages revealed higher phosphorylation of signal transducer and activator of transcription in response to interferon gamma and a rapid decline in the level of inhibitory kappa B-alpha protein induced by lipopolysaccharide in B10R macrophages compared to B10S. Altogether, our results demonstrate a link between allelic expression of the Bcg/Nramp1 gene and alterations in several macrophage signaling pathways, and support the hypothesis that the allelic expression of Bcg/Nramp1 may be functionally linked to resistance to infectious disease and, inversely, to autoimmune disease susceptibility.

Proteome study of colorectal carcinogenesis.

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.

The alveolar septal thickness and type II pneumocytes number in irradiated lungs, time expression and the effect of pentoxifylline.

PURPOSE: We studied the relationship between type II pneumocytes number and alveolar septal thickness during different time after sublethal whole-thorax irradiation of rats and we investigated the influence of pentoxifylline (TNF-alpha inhibitor). MATERIALS AND METHODS: Wistar rats were exposed to 15 Gy thoracic irradiation and pentoxifylline (35 mg/kg) twice a week. Lungs were examined histologically and immunohistochemically at intervals ranging from 1-12 weeks and alveolar septal thickness, number of type II pneumocytes (identified by immunoreactivity for cytokeratin 18), and neutrophile granulocytes were counted. RESULTS: Significant increase of alveolar septal thickness and type II pneumocytes depletion 3 weeks after irradiation were found. Correlation of these markers was r = -0.759. Pentoxifylline significantly inhibits increased alveolar septal thickness without the influence on type II pneumocytes number. Neutrophil penetration started 5 weeks after irradiation in non-treated animals, 8 weeks after irradiation in PTX-treated rats. CONCLUSIONS: We suggest that pneumocytes depletion is linked to increased vascular permeability, and pentoxifylline therapy does not influence on pneumocytes kinetics after irradiation.

Tick salivary gland extract inhibits killing of Borrelia afzelii spirochetes by mouse macrophages.

Salivary gland extract (SGE) from Ixodes ricinus ticks inhibited the killing of Borrelia afzelii spirochetes by murine macrophages. SGE also reduced the production of two major defense molecules of phagocytes, superoxide and nitric oxide. It is likely that the suppression of macrophage microbicidal mechanisms contributes to the inhibitory effect of tick saliva on the killing of B. afzelii spirochetes, thus facilitating the transmission of this important pathogen.

Apoptosis and bcl-2 expression in irradiated lungs and the effect of pentoxifylline.

We measured number of bcl-2, apoptotic, neutrophil, and surfactant apoprotein D (SP-D) positive cells in irradiated rat lungs during different time points after the sublethal whole-thorax irradiation of rats. We also investigated the influence of pentoxifylline (PTX) therapy on these markers. Wistar rats were given 15 Gy thoracic irradiation and PTX (35 mg/kg) twice a week. Animals were examined histologically and imunohistochemically at intervals from 1-12 weeks. In non-treated rats compared with treated rats, bcl-2 expression was significantly inhibited from 4 weeks after irradiation. A higher apoptosis presence in non-treated rats from 4 weeks was found and apoptosis development in PTX-treated animals was delayed and started 8 weeks after irradiation. Similar differences were measured during neutrophil granulocytes examination. Neutrophil penetration in non-treated rats was found 5 weeks after irradiation in contrast to the RP onset of PTX-treated animals 8 weeks after irradiation. The number of SP-D positive cells in non-treated rats observed until 5 weeks after irradiation was higher than in the control group. PTX-treated animals expressed higher number of SP-D positive cells during the whole experiment than the control group. We suggest that apoptosis is linked to neutrophil granulocyte actions during the RP onset and that PTX-therapy causes diminished inflammation development.

The immunomodulatory effect(s) of lead and cadmium on the cells of immune system in vitro.

A number of studies documented that the heavy metals are not only toxic for the organisms but they may modulate immune responses. The immunomodulatory activity was proved in several in vivo and in vitro model systems. In the current study, immunomodulatory activities of lead and cadmium are presented. The viability of both lymphocytes and macrophages was affected by heavy metals in a dose- and time-dependent manner. In the case of lead, the depression of N-oxide production closely correlated with increased blast transformation of spleen cells induced by concanavalin A (ConA). On the contrary, cadmium suppressed the production of N-oxides but stimulated significantly the proliferation of spleen cells. The production of cytokines by lymphocytes and macrophages was dependent on the in vitro model used. Generally, the treatment of macrophages with lead results in disregulation of the production of proinflammatory cytokines [tumour necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha) and interleukin 6 (IL-6)] and preferential production of Th1 type of cytokines (IFN-gamma and IL-2). Cadmium seemed to trigger the Th2 cytokine regulatory pathway [interleukin 4 (IL-4), interleukin 10 (IL-10)]. The results suggest the metal-induced changes in immunoregulatory mechanism of host with potentially severe clinical consequences.

Influence of the bcg locus on natural resistance to primary infection with the facultative intracellular bacterium Francisella tularensis in mice.

The implication of the Bcg locus in the control of natural resistance to infection with a live vaccine strain (LVS) of the intracellular pathogen Francisella tularensis was studied. Analysis of phenotypic expression of natural resistance and susceptibility was performed using mouse strains congenic at the Bcg locus. Comparison of the kinetics of bacterial colonization of spleen showed that B10.A.Bcg(r) mice were extremely susceptible during early phases of primary sublethal infection, while their congenic C57BL/10N [Bcg(s)] counterparts could be classified as resistant to F. tularensis LVS infection according to the 2-log-lower bacterial CFU within the tissue as long as 5 days after infection. Different phenotypes of Bcg congenic mice were associated with differential expression of the cytokines tumor necrosis factor alpha, interleukin-10, and gamma interferon and production of reactive oxygen intermediates. These results strongly suggest that the Bcg locus, which is close or identical to the Nramp1 gene, controls natural resistance to infection by F. tularensis and that its effect is the opposite of that observed for other Bcg-controlled pathogens.

Protein abundance alterations in matched sets of macroscopically normal colon mucosa and colorectal carcinoma.

Our current results, aimed at the detection of protein abundance alterations that could be associated with the process of colon tumorigenesis, are summarized. The matched sets of macroscopically normal colon mucosa and colorectal carcinoma were examined by a one- or two-dimensional electrophoretic approach and proteins were identified using immunoblotting or mass spectrometry. The following results were observed: The levels of liver fatty acid-binding protein, actin-binding protein/smooth muscle protein 22-alpha and cyclooxygenase 2 were downregulated in colorectal carcinoma compared to normal colon mucosa. Conversely, the expression of a novel variant of heat shock protein70 and several members of the S100 protein family of calcium-binding proteins (two isoforms of S100A9, S100A8, S100A11 and S100A6) were upregulated in transformed colon mucosa. Despite the variations of the levels of expression of given protein among analyzed samples, all quantitative changes were found to be statistically significant (Mann-Whitney test assuming p < or = 0.05). We conclude that the proteomic approach is useful for the study of complex biological events underlying the process of colorectal tumorigenesis.

[Immunostimulating activity of vaccines in the treatment of recurrent urinary tract infections. I]. / Immunostimulacní aktivita vakcíny pro lécbu recidivujících uroinfekcí. I.

The authors inform about the immunomodulatory properties of the vaccine URVAKOL aimed for the treatment of recidiving urinary infections. The results of immunostimulatory activity of the preparation and its effects on cellular and humoral immunity in mice following intraperitoneal administration of the vaccine are presented. The vaccine markedly increases cytotoxic activity of adhering peritoneal cells and has protective effects in model infection induced by intracellular pathogen Francisella tularensis (strain 15 L). (Tab. 6, Fig. 6, Ref. 9.)

Modulation of signal transduction pathways and global protein composition of macrophages by ionizing radiation.

It is assumed that the exposure of cells to ionizing radiation modulates their signal transduction pathways, which then govern the early and late radiation-induced alterations in gene expression. In this study we tested the effects of low doses of X-irradiation on the cell signaling and global protein composition of an HL-60 human promyelocytic leukemia cell line differentiated along a macrophage-like cell pathway by 4beta-phorbol-12-myristate-13-acetate (PMA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting of anti-phosphotyrosine immunoprecipitates, we found radiation-induced changes in the level of phosphorylation of proteins with molecular masses of 45 and 48 kDa, but in the most intensively stained area, ranging from 54 to 60 kDa, no alterations were observed. When two-dimensional electrophoresis (2-DE) immunoblotting was applied, only proteins from this heavily stained region were visualized and in addition the evident differences in tyrosine phosphorylated protein patterns between nonirradiated and irradiated cells were found in this area. Furthermore, the immunostaining of extracellular signal-regulated kinase 2 (ERK2) which did not prove its tyrosine phosphorylation demonstrated the existence of several ERK2 charge isoforms showing differential expression after X-irradiation. Comparing the whole protein profiles we found after the simultaneous quantitation of 1000 matched spots two proteins whose expression was regulated in an opposite manner in nonirradiated and X-irradiated cells. The quantities of both spots showed increases or decreases by a factor of 2 or more between irradiated and nonirradiated samples and both these changes were statistically significant (P<0.05).

The analysis of S100A9 and S100A8 expression in matched sets of macroscopically normal colon mucosa and colorectal carcinoma: the S100A9 and S100A8 positive cells underlie and invade tumor mass.

The expression of calcium-binding protein S100A9 was investigated in 23 matched sets of colorectal carcinoma and normal colon mucosa using two-dimensional gel electrophoresis. We found that, from a group of 23 patients, the level of S100A9 protein, in comparison with matched normal colon mucosa, was significantly increased in malignant tissues of 16 patients (70%). Furthermore, an additional protein, identified by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) as S100A8, exhibited an increased expression in the same specimens of malignant tissues as the S100A9 protein. The immunohistological analysis revealed the accumulation of S100A9 positive cells, macrophages and polymorphonuclear leukocytes along the invasive margin of colorectal carcinoma. The S100A8 protein was found to be produced in the same location. The possible participation of both proteins and, especially, its heterodimeric complex calprotectin in colorectal carcinoma regression could be taken into account.

[Immunostimulatory activity of the vaccine used in the treatment of recurrent urinary infections. II]. / Imunostimulacní aktivita vakcíny pro lécbu recidivujících uroinfekcí. II.

The authors describe on the immunostimulatory properties of the vaccine URVAKOL aimed for the treatment of recurrent urinary infections. Detection of immunostimulatory activity of the preparation and its effects on the humoral and cellular immunity were performed after oral administration of the preparation. Important was the evidence of nonspecific immunity of mice against intracellular pathogen Francisella tularensis induced with URVAKOL strain 15L. (Tab. 4, Fig. 1, Ref. 8.)

Production of stress-inducible form of heat-shock protein 70 in mouse peritoneal adherent cells after in vivo infection by Francisella tularensis.

Heat-shock proteins (hsp) are ubiquitously produced molecules which participate in the protection of cells from environmental perturbation. Moreover, the members of the heat-shock protein 60 (hsp60) and 70 (hsp70) families play an important role in pathogen-host interactions. We studied in vivo production of the 70-kDa heat-shock proteins in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with an attenuated vaccine strain (LVS) of Francisella tularensis. We found a differential production of a highly stress-inducible member of the hsp70 family, designated hsp72, in three inbred strains of mice exhibiting either resistance or susceptibility to F. tularensis LVS infection. Whereas in tularemia-resistant mice hsp72 was even expressed in PEC without injection of bacteria and its production further increased on day 3 and slowly declined on days 5 and 7 after injection, in susceptible mice hsp72 production was highly inducible and restricted only to day 3 after in vivo infection. Further analysis of hsp72 expression revealed intracellular hsp72 accumulation and its preferential production by peritoneal adherent cells.

[Tularemia, "volume" two]. / Tularemie, "kniha" druhá.

As a continuation of Libich's monograph (Tularemie. Prague, Avicenum 1981) the author presents findings assembled in experiments pertaining to postinfectious immunity on a model of intracellular infection with the microorganism Francisella tularensis.

Natural resistance to intracellular parasites: a study by two-dimensional gel electrophoresis coupled with multivariate analysis.

Natural resistance to Mycobacterium bovis bacillus Calmette-Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis. When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.